Development of a 64Cu-labeled CD4+ T cell targeting PET tracer: evaluation of CD4 specificity and its potential use in collagen-induced arthritis

Background CD4+ T cells are central inflammatory mediators in the pathogenesis of autoimmune rheumatoid arthritis (RA), as they are one of the dominating cell types in synovial inflammation. Molecular imaging of CD4+ T cells has potential role for early detection and monitoring of RA. Here, we developed a new radiotracer for in vivo immunoPET imaging of murine CD4+ T cells and tested it in the collagen-induced arthritis (CIA) mouse model of human RA. Results The tracer, [64Cu]Cu-NOTA-CD4-F(ab)’2 ([64Cu]Cu-NOTA-CD4), was generated from F(ab)’2 fragments of R-anti-mouse CD4 antibodies conjugated to the 2-S-(isothiocyanatbenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (p-SCN-Bn-NOTA) chelator and radiolabeled with copper-64. Accumulation of the tracer and isotype control was evaluated in the CIA model and mice receiving whole-body irradiation (WBI) (5 Gy). The potential of [64Cu]Cu-NOTA-CD4 for response assessment was evaluated in CIA induced mice treated with dexamethasone (DXM). Imaging data were compared with flow cytometry and immunohistochemistry (IHC) of inflammatory cells including CD4+ T cells. [64Cu]Cu-NOTA-CD4 showed increased accumulation in T cell-rich tissues compared with isotype control (p < 0.0001). In addition, reduced accumulation of [64Cu]Cu-NOTA-CD4 was observed in T cell-depleted tissue (p < 0.0001). Flow cytometry and IHC confirmed the increased infiltration of CD4+ T cells in CIA mice. Conclusions We developed and evaluated a new radiotracer, [64Cu]Cu-NOTA-CD4, for immunoPET imaging of murine CD4+ T cells. [64Cu]Cu-NOTA-CD4 was successfully synthesized by F(ab)’2 fragments of R-anti-mouse CD4 antibodies conjugated to a chelator and radiolabeled with copper-64. We found that our novel CD4 PET tracer can be used for noninvasive visualization of murine CD4+ T cells. Supplementary Information The online version contains supplementary material available at 10.1186/s13550-022-00934-7.


Introduction
Rheumatoid arthritis (RA) is an autoimmune, inflammatory disease with an estimated prevalence of 1% worldwide [1]. It is a long-term, progressive disorder that can result in chronic inflammation and articular destruction of multiple joints. The pathogenesis involves T cell-mediated recognition of arthritis-specific autoantigens through major histocompatibility complex class II-mediated presentation [2,3]. A variety of immune cells play a role in the hyperplasia progression of synovial membranes (synovium), cartilage degeneration and bone tissue destruction [4]. However, CD4 + T cells are key mediators of tissue damage in the joints and play a crucial role in disease initiation [5,6]. If left undiagnosed, untreated or unresponsive to therapy, inflammation and joint destruction will lead to loss of physical function and have severe consequences for the patient including difficulties and potentially inability in maintaining daily living. Therefore, early diagnosis and treatment initiation is of paramount importance.
The diagnosis of RA mainly depends on the clinical phenotype and serology testing. However, as RA is a progressive disease, advanced disease can be assessed by imaging including X-rays, ultrasound and MRI [7]. Nevertheless, these techniques have low sensitivity of detecting articular changes at an earlier time point. The modalities are not well established in the clinic and a need for new methods for earlier detection and confirmation of cases occur. The clinical need is not limited to consistent, predictive biomarkers of prognosis, but also of therapeutic response markers in order to improve patient compliance [8]. Here, we focus on the potential advantages of using immunoPET imaging to detect the inflammatory CD4 + T cell response in the collageninduced arthritis (CIA) model, a mouse model of RA. ImmunoPET has mostly been used in oncology for tumor associated antigens but has previously been expanded to several indications including imaging of infections and inflammatory diseases [9]. Using molecular imaging to target specific inflammatory biomarkers allows for visualization of cells and molecules that are involved in the process. Imaging also allows for assessment of localized inflammatory activity, which is highly relevant in RA. Finally, the early diagnosis and treatment initiation of patients with inflammatory conditions is crucial, as it can prevent irreversible tissue damage.
As RA is a T cell-mediated disease, we hypothesized that application of murine T cell-specific antibody F(ab)′2 fragments could be useful to track T cell migration in the CIA model [10]. In the present study, we therefore developed a new radiotracer for noninvasive immun-oPET of murine CD4 + T cells. F(ab)′2 fragment targeting murine CD4 + T cells was conjugated to the chelator 2-S-(isothiocyanatbenzyl)-1,4,7-triazacyclononane-1,4,7triacetic acid (p-SCN-Bn-NOTA) and radiolabeled with copper-64. [ 64 Cu]Cu-NOTA-CD4-F(ab)'2 ([ 64 Cu] Cu-NOTA-CD4) was evaluated as a predictive imaging biomarker in the CIA model. First, the optimal imaging time point was determined in a longitudinal biodistribution study. Next, the uptake of [ 64 Cu]Cu-NOTA-CD4 following systemic administration of dexamethasone (DXM) was assessed in inflamed arthritic lesions. Finally, target-specific affinity of [ 64 Cu]Cu-NOTA-CD4 and the isotype control radiotracer [ 64 Cu]Cu-NOTA-IgG2b-F(ab)'2 ([ 64 Cu]Cu-NOTA-IgG2b) was evaluated. To validate the findings using other methods, infiltration of CD4 + T cells in the inflamed joints was assessed by flow cytometry and immunohistochemistry (IHC) and compared with CD4 PET imaging.

Mice
Female DBA/1JRj mice (7 weeks) and C57BL/6JRj (7 weeks) were purchased (Janvier, Le Genest-Saint-Isle, France) and housed in groups of 4-8 mice in individually ventilated cages under standardized lighting conditions. Mice were fed pathogen-free food and water ad libitum. All animal experiments were conducted under protocols approved by the National Animal Experiments Inspectorate under the license number 2016-15-0201-00920.

Collagen-induced arthritis (CIA)
Mice were immunized subcutaneously at the base of the tail (100 µL) with chicken CII (Chondrex, Redmond, WA, USA) emulsified in complete Freund's adjuvant (Sigma-Aldrich, St. Louis, MO, USA). Three weeks after initial immunization, mice received a booster immunization with CII emulsified in incomplete Freund's adjuvant (Sigma-Aldrich, St. Louis, MO, USA) (100 µL) [11]. CII was dissolved O/N at 4 °C in 10 mM acetic acid. Mice were anesthetized in 3-4% sevoflurane in 65% N 2 and 35% O 2 during both injections. Signs of arthritis in the paws appeared approximately four weeks after initial immunization. Each paw was visually scored three times a week on a scale from 0 to 4. The following criteria were used: 0: normal paw, 1: one toe inflamed and swollen, 2: more than one toe, but not entire paw, inflamed and swollen or mild swelling of entire paw, 3: entire paw inflamed and swollen and 4: very inflamed and swollen paw or ankylosed paw [12]. For ethical purposes, a mean score of 2.5 for all paws was considered maximum per animal. Mice were euthanized if exceeding the mean score. Data are presented as the mean score of all four paws. In case of lameness, score ≥ 2, the mice were treated with daily analgesia (buprenorphine, 1 mg/kg, intraperitoneal (i.p.)). For therapeutic assessment, the CIA mice were treated daily with DXM for 7 days starting on day 28 after initial immunization [13][14][15]. DXM was administered as i.p. injections in a total volume of 200 µL/mouse (1 mg/kg in NaCl).
The immunoreactivity of anti-CD4-F(ab)'2 fragments following radiolabeling was assessed according to the Lindmo assay [17]. Increasing concentrations of CD4 + cells (2.5 × 10 6 -4 × 10 7 cells/mL) were incubated with 1 nM [ 64 Cu]Cu-NOTA-CD4 for 3 h at 4 °C. Cells were centrifuged at 500 g for 5 min and the supernatants and pellets counted in a gamma counter (Wizard 2 , Perki-nElmer, Massachusetts, USA). Cell-associated radioactivity was calculated as the ratio of cell-bound radioactivity to the total amount of added radioactivity.
The affinity of radiolabeled anti-CD4-F(ab)'2 was assessed by a saturation binding assay. T cells were harvested as described above, added in triplicates (2 × 10 4 cells) to a MultiScreenHTS BV Filter Plate 1.2 µm (#MSBVN1250, Merck Millipore) and washed twice in PBS. Eight different concentrations of [ 64 Cu]Cu-NOTA-CD4 [range: 60-0.04 nM] in PBS supplemented with 1% bovine serum albumin (BSA) were added the wells. The plate was incubated for 4 h at 4 °C. After incubation, the plate was washed 3 times in PBS with 1% BSA using a vacuum manifold (Macherey-Nagel, Fisher Scientific). The plastic cover was removed from the plate bottom and the plate dried in a heat cabinet. The dry filters were transferred to counting tubes and counted in a gamma counter.

Imaging analysis
Image analysis was performed using the Inveon ® Research Workstation software (Siemens Medical Systems, PA, USA). A CT-based region of interest (ROI) tool was used to carefully draw around each carpal and tarsal joint, forming four ROIs per mouse. ROIs were also drawn over the heart, kidney, spleen, thymus, liver and thigh muscle. The activity in the blood was calculated as 20% maximum activity in the heart. The uptake of [ 64 Cu]Cu-NOTA-CD4 and [ 64 Cu]Cu-NOTA-IgG2b was quantified as percent injected dose per joint (%ID/joint) and maximum percent injected dose per gram tissue (%Max ID/g) assuming a soft tissue density of 1 g/cm 3 . Target-to-blood ratios of [ 64 Cu]Cu-NOTA-CD4 uptake were calculated as maximum uptake (%Max ID/g) in the most affected joints (score 3) divided by mean uptake in the blood (%ID/g) to determine optimal scanning time. Moreover, joint-to-blood ratios were calculated for the DXM assessment.

Immunohistochemical analysis
CIA and control mice were euthanized by cervical dislocation and the carpal and tarsal joints were isolated. Joints were decalcified for three weeks in 10% formic acid in buffered formaldehyde 4% followed by preparation in Shandon Excelsior AS Tissue Processor (Thermo Fisher Scientific, Waltham, MA, USA) O/N and embedded in paraffin. Paraffin-embedded joints were sectioned at 4 µM and dewaxed through xylene to tap water. For antigen retrieval, sections were boiled in microwave for 15 min in 10 mmol citrate buffer (pH 6) and pre-incubated in 2% bovine serum albumin (BSA) for 10 min followed by incubation with primary recombinant anti-CD4 antibody (#ab183685, Abcam, Cambridge, UK) at 1:500 dilution in 2% BSA O/N at 4 °C. Sections were incubated for 40 min with biotinylated secondary goat-antirabbit IgG antibody (BA-1000, Vector Laboratories, Burlingame, CA, USA) at 1:200 dilution. Afterward, 3% hydrogen peroxide blocked the endogenous peroxidase. To amplify the reaction, sections were incubated with Avidin and Biotinylated horseradish peroxidase macromolecular Complex (ABC-Elite) (PK-6100, Vector Laboratories) for 30 min. Finally, the reaction was developed by the use of 3,3-diaminobenzidine (SK-4100, Vector Laboratories) for 15 min and counterstaining was performed with Mayer's Hematoxylin Solution (Sigma-Aldrich, St. Louis, MO, USA). All procedures were performed at RT if not stated otherwise. Sections were stained in the same analysis. Images were taken using an Olympus BX51 microscope with a XC-10 camera.

Flow cytometry
Joint-infiltrating cells were isolated by a procedure adapted from [18]. Briefly, the carpal and tarsal joints were isolated. Skin and fur were removed, and joints were mechanically dissociated using surgical scissors followed by digestion in RPMI 1640 supplemented with type 1-S hyaluronidase from bovine testes (2.4 mg/mL) (Sigma, St. Louis, MO, USA), collagenase VIII (1 mg/mL) (Sigma), 10 mM HEPES, 50 units/mL penicillin and 50 µg/mL streptomycin for 1 h at 37 °C in a shaking water bath. Digested joints were passed through a 70 µm cell strainer.
Processed cells were blocked with Fc-block (clone 2.4G2) (BD Biosciences, San Jose, CA, USA) in FACS buffer (PBS + 0.5% BSA + 0.1% NaN3 + 2 mM EDTA) for 5 min on ice. Fc-blocked samples were stained for 30 min on ice in a master mix of FACS buffer, brilliant stain buffer, amine reactive dye (eFluor780 viability dye) (Thermo Fisher Scientific, Waltham, MA, USA) and the following antibodies

Statistical analysis
One-way analysis of variance with Tukey's post hoc test was used to assess statistically significant differences between groups. p values < 0.05 were considered statistically significant. Prism 8.0c (GraphPad Software, La Jolla, CA, USA) was used for all statistical analysis.
SDS-PAGE analysis by Coomassie staining and radiography of the same gel, confirmed the digestion efficiency and revealed [ 64 Cu]Cu-NOTA-CD4-F(ab)'2 at 100 kDa (lane 3) with no presence of digested fragments or fulllength antibody in the final product. In addition, the fulllength anti-mouse CD4 antibody (IgG) at 150 kDa (lane 1), the digested antibody (lane 2) with heavy chains (Hc) and light chains (Lc) are shown (Fig. 1d). The full-length SDS-PAGE stained is shown in Additional file 1: Fig S1a, and radiography of the full-length gel (only exposed once) is shown in Additional file 1: Fig. S1b.

Longitudinal PET evaluation of [ 64 Cu]Cu-NOTA-CD4 in arthritic lesions
Mice immunized with chicken CII emulsion in CFA (day 0) followed by a booster immunization with the emulsion in IFA (day 21) developed macroscopic joint inflammatory including swelling and erythema of carpal and tarsal joints starting on day 28 after initial immunization. Monitoring of animals was performed on day 28, 32, 33 and 35 with mean CIA score per animal of 0.83 ± 0.24, 1.29 ± 0.16, 1.54 ± 0.18 and 1.63 ± 0.18, respectively (Fig. 2a, b).
The   a significantly increased activity in the most affected joints (score 3) 1 h (1.01 ± 0.15% ID/joint, p < 0.0001), 4 h (1.04 ± 0.17% ID/joint, p < 0.0001) and 24 h (0.33 ± 0.07% ID/joint, p = 0.0052) p.i. in comparison with control mice. The uptake in the most pronounced arthritic joints had a drop after the two initial time points in comparison with the two latter time points. At the 44 h p.i. imaging time point the activity was 0.18 ± 0.08% ID/joint. Moreover, the maximum uptake within the most affected joints (score 3) was 15.5 ± 0.9, 16.8 ± 1.8, 11.7 ± 1.3 and 15.9 ± 4.4% ID/g for the 1, 4, 24 and 44 h time points, respectively. The maximum uptake in joints with score 3 was significantly increased at all time points compared with control mice (1 h: p = 0.0079; 4 h: p = 0.0003; 24 h: p = 0.0003 and 44 h: p < 0.0001) (Fig. 2c).
Representative PET/CT images of a CIA mouse at 1, 4, 24 and 44 h p.i. of [ 64 Cu]Cu-NOTA-CD4 are shown in Fig. 2d. The presented mouse had obvious clinical symptoms of arthritis including swelling and erythema of the left carpal and right tarsal joint, which mutually scored 3. The increased activity in these arthritic joints is visualized at all time points. Clearance of the background can also be seen throughout the temporal imaging.
In vivo biodistribution profile of [ 64 Cu]Cu-NOTA-CD4 was performed in the organs of interest. The organ of interest was spleen and thymus, since they are lymphoid organs, blood for circulation time and liver and kidney for excretion. The organ of interest showed high accumulation in blood, kidneys, spleen, liver and thymus at the initial time points (1 and 4 h) followed by a decrease at the late time points (24 and 44 h) (Fig. 3). The activity in the blood measured as 20% maximum activity in ROIs drawn over the heart decreased between the initial and late time point. Specifically, blood activity decreased from 21.6 ± 0.5 and 14.7 ± 0.9% ID/g (1 and 4 h p.i.) to 2.1 ± 0.3 and 1.6 ± 0.2% ID/g (24 and 44 h p.i.). This demonstrates the renal and hepatic clearance of tracer from blood.
Joint-to-blood ratios of [ 64 Cu]Cu-NOTA-CD4 uptake in most affected joints (score 3) significantly increased throughout the imaging time points (1 to 44 h) (p = 0.0071) ( Table 1). Based on these results, 24 h p.i. was chosen as PET imaging time point for the therapy assessment. This was chosen although the highest jointto-blood ratio of [ 64 Cu]Cu-NOTA-CD4 was detected 44 h p.i.
[ 64 Cu]Cu-NOTA-CD4 accumulation correlates with arthritic inflammation levels in mice DXM, a corticosteroid with known anti-inflammatory effects, was used to evaluate the potential of [ 64 Cu] Cu-NOTA-CD4 to differentiate between changes in CD4 + T cells in both systemically DXM treated and untreated arthritic lesions. The study was performed according to the setup in Fig. 4a. The mice were monitored on day 26, 28, 29, 30, 31, 33 and 35, and the mean CIA score for mice with arthritic lesions was 0.10 ± 0.04, 0.41 ± 0.12, 0.75 ± 0.16, 0.55 ± 0.09, 0.88 ± 0.18, 0.80 ± 0.11 and 0.93 ± 0.12, respectively (Fig. 4b). Treatment was initiated on day 28 after initial immunization. At this time point, 5 out of 12 mice (41.7%) showed initial signs of disease with a mean CIA score of 0.21 ± 0.11. Mice displayed an overall decrease in CIA score after receiving DXM (Fig. 4c).
The in vivo [ 64 Cu]Cu-NOTA-CD4 PET activity was quantified 24 h p.i. in DXM and untreated CIA mice.

[ 64 Cu]Cu-NOTA-CD4 and isotype control accumulation in arthritic joints and T cell-rich tissues
For evaluation of in vivo specific targeting of [ 64 Cu] Cu-NOTA-CD4, a control isotype imaging study was conducted in CIA mice. This was performed to determine if the activity in inflamed joints was due to interaction between [ 64 Cu]Cu-NOTA-CD4 and CD4 + T cells. The in vivo [ 64 Cu]Cu-NOTA-IgG2b uptake was quantified 24 h p.i. as previous. Results revealed increased [ 64 Cu]Cu-NOTA-IgG2b activity in joints with high arthritis score (score 3: 0.80 ± 0.12% ID/joint and 10.88 ± 0.39% Max ID/g) in comparison with control mice (0.15 ± 0.04% ID/joint and 4.69 ± 0.66% Max ID/g) (p < 0.0001) (Fig. 5a).

Lymphodepleting whole-body irradiation reduces [ 64 Cu] Cu-NOTA-CD4 but not isotype control activity in the spleen
Due to the high activity of [ 64 Cu]Cu-NOTA-CD4 in the spleen and thymus compared with [ 64 Cu]Cu-NOTA-IgG2b, a biodistribution study in WBI and non-irradiated C57BL/6 mice was conducted to test for tracer specificity to CD4 + T cells. A significantly lower accumulation of [ 64 Cu]Cu-NOTA-CD4 was observed in WBI (12.12 ± 1.20% ID/g) compared with non-irradiated control mice (15.54 ± 0.46% ID/g) (p < 0.0001). This indicates a depletion of CD4 + cells in the spleen following a radiation dose of 5 Gy (Fig. 6a). A representative [ 64 Cu] Cu-NOTA-CD4 PET/CT image of a WBI mouse is displayed in Fig. 6b.

Discussion
In this study, we developed and evaluated a novel radiotracer, [ 64 Cu]Cu-NOTA-CD4, for immunoPET imaging of murine CD4 + T cells. [ 64 Cu]Cu-NOTA-CD4 was successfully synthesized by F(ab)'2 fragments of R-anti-mouse CD4 antibodies conjugated to a chelator and radiolabeled with copper-64. We found that [ 64 Cu] Cu-NOTA-CD4 could be used for noninvasive visualization of CD4 + T cells. Importantly, [ 64 Cu]Cu-NOTA-CD4 accumulates in T cell-rich tissues compared with the control isotype tracer, [ 64 Cu]Cu-NOTA-IgG2b, and target-specific affinity was also supported by reduced uptake of [ 64 Cu]Cu-NOTA-CD4 in T cell-depleted tissue.
Our findings showed that [ 64 Cu]Cu-NOTA-CD4 accumulates in tissues with high level of CD4 + T cells and can follow stimuli affected T cell levels. Imaging of CD4 + T cells has an increasingly important role reflecting the pivotal role of CD4 + T cells in many inflammatory diseases including RA, inflammatory bowel disease and multiple sclerosis [6,19,20]. These conditions are CD4 + T cell driven, and CD4 + T cells play a crucial role in disease initiation. Previously, several monoclonal antibodies and their fragments have been radiolabeled and investigated for preclinical immunoPET utilization [9]. Researchers have especially taken advantage of immunoPET within the field of oncology for tumor associated antigens, but this has expanded to several indications including inflammation imaging [21].
In our studies, we used antibody F(ab)'2 fragments with two antigen-binding regions, as these have great potential as diagnostic components. Previous studies also demonstrated the use of radiolabeled antibody fragments for imaging of inflammation. T cell tracer of GK1.5 cys-diabody (cDb), an anti-mouse CD4 antibody fragment, radiolabeled with Zirconium-89 for PET imaging [22]. Here, [ 89 Zr]Zr-malDFO-GK1.5 cDb detected CD4 + T cells in the distal colon of dextran sulfate sodium-induced colitis in a mouse model of inflammatory bowel disease. Beneficially, antibody fragments have shorter circulation times (hours), deeper tissue penetration, allow imaging the same day and can therefore be radiolabeled with shorter-lived PET radionuclides, e.g., flourine-18 (t ½ = 109.7 min) and gallium-68 Fig. 7 CD4 + T cell infiltration in arthritic joints. a Representative immunohistochemical images of CD4 infiltration in a joint with collagen-induced arthritis (CIA) (score 3; A: upper panels) and in a joint from a control mouse (A: lower panels). Mice with CIA were scored according to degree of inflammation (swelling and erythema) in each paw on a scale from 0 to 4. b Flow cytometric analysis with infiltration of CD4 + T cells and neutrophils as percent of viable cells in the carpal and tarsal joints from control, dexamethasone (DXM) treated and untreated CIA mice. Each point represents a joint. Gating strategy is shown in Additional file 1: Fig. S2. Data are presented as mean ± SEM and are pooled from two independent experiments with similar results. The significance level is indicated by asterisks (*). p < 0.01 (**), p < 0.001 (***) and p < 0.0001 (****) (t ½ = 68 min). However, using positron emitters with a longer physical half-lives such as copper-64 (t ½ = 12.7 h) enable immunoPET imaging the following day (24 h) [23]. In our longitudinal biodistribution study, the optimal imaging time point of 24 h p.i. was determined.
We also showed increased accumulation of [ 64 Cu] Cu-NOTA-CD4 in the spleen and thymus compared with the isotype, [ 64 Cu]Cu-NOTA-IgG2b. For further elucidation, splenic CD4 + T cell depletion was conducted by WBI in mice of 5 Gy. Accordingly, the uptake of [ 64 Cu] Cu-NOTA-CD4 significantly decreased in irradiated mice compared with non-irradiated mice, supporting the target-specific binding of [ 64 Cu]Cu-NOTA-CD4. Similar depletion has previously been shown by Wei et al. [24], who significantly reduced number of CD4 + T cells in the spleen two days after local tumor irradiation (8.5 Gy daily) of female C57BL/6JRj mice. Likewise, Li et al. [25] showed that 5 Gy of WBI significantly reduced the number of CD4 + T cells after seven days in the spleen. This clearly indicates that [ 64 Cu]Cu-NOTA-CD4 targets CD4 + T cells but also clearly demonstrates the important differences between inflammatory and healthy tissues including vascular changes, increased blood flow, poor lymphatic drainage and edema [26].
Our findings in arthritic joints highlight an important challenge for large radiolabeled antigen-binding molecules, including the F(ab)´2 fragment. The results indicate that pathophysiological changes in lesions with pronounced inflammation stimulate tracer extravasation and retention. This may provide a situation where tracer activity and retention are the result of both target (ligand)-specific binding and an inflamed enhanced permeability and retention (EPR)-like effect to some degree. We have previously demonstrated that radiolabeled liposomes accumulate at very high levels in different types of inflammatory lesions [27]. This accumulation may illustrate the situation in cancerous tissues, where liposomes accumulate by the well-described EPR effect [28,29]. The EPR effect results in non-specific extravasation of macromolecules from the blood stream via fenestration of the endothelial lining. The molecules are then retained from leaving the cancerous tissue [30]. In the light of the observed non-specific accumulation of radiolabeled F(ab)'2 fragments in severe inflammatory lesions, we therefore speculate that a comparable challenge may exist. In addition, we speculate that unspecific uptake of the tracer could be caused by increased phagocytic activity within the arthritic lesions with pronounced inflammation. This approach is also supported by the ex vivo analysis of the inflamed joints by flow cytometry, which showed increased number of neutrophils in joints with pronounced inflammation compared with healthy control and DXM-treated joints. Careful assessment of accumulation specificity and retention of F(ab)'2 fragments and comparable sized radiotracers are therefore warranted to secure validity.
Nevertheless, our results only revealed increased accumulation of isotype control, [ 64 Cu]Cu-NOTA-IgG2b, in joints with pronounced arthritis. No increased uptake in tissues with mild arthritis and healthy tissue was observed, indicating specific targeting of our CD4 PET tracer, [ 64 Cu]Cu-NOTA-CD4, in lesions with mild inflammation. Therefore, we speculate that the specificity of our CD4 PET tracer toward the increased number of CD4 + cells is sufficient to exceed the passive targeting by the EPR effect or potentially uptake by phagocytosis in joints with mild inflammation. Kristensen et al. [16] previously tested similar CD4 + -and CD8 + -specific PET tracers but radiolabeled with zirconium-89, [ 89 Zr]Zr-DFO-CD4 and [ 89 Zr]Zr-DFO-CD8a, in several preclinical mouse models of cancer. Here, tracers were used to phenotype tumors at an early stage and also allow following the treatment response. The possibility of unspecific tracer uptake in the heterogenic tumor microenvironment caused by the EPR effect was also discussed. Careful assessment and inclusion of isotype controls are therefore of great importance for indications beyond inflammation. Altogether, this indicates that the use of our CD4 PET tracer, [ 64 Cu] Cu-NOTA-CD4, should be centered toward following the response to treatment and detecting potential recurrence rather than initial diagnosis. It could be argued that imaging of inflammation could easier be done with [ 18 F]-FDG PET. However, it has previously been shown that not only is the uptake of [ 18 F]-FDG low in arthritis but also that it seems not to correlate with severity of arthritis, i.e., arthritis score [31].
Radionuclide imaging of CD4 in arthritis adds to imaging of fibroblasts activating protein and F4/80 receptor positive macrophages [32].

Conclusion
We developed and evaluated a novel radiotracer, [ 64 Cu] Cu-NOTA-CD4, for immunoPET imaging of murine CD4 + T cells. [ 64 Cu]Cu-NOTA-CD4 was successfully synthesized by F(ab)'2 fragments of R-anti-mouse CD4 antibodies conjugated to a chelator and radiolabeled with copper-64. We found that our novel CD4 PET tracer can be used for noninvasive visualization of murine CD4 + T cells and may be used for noninvasive studies of inflammatory conditions including RA.